Fig. 3: MDA5 expression leads to pluripotency transcription factor downregulation and innate immune signalling.

a Differential accessible sites (ATAC-seq) for WT and MDA5 expressing cells after 24 h doxycycline treatment. Data represent the average of two biological replicates. b KEGG pathway analyses of genes associated with differential ATAC peaks, with the more accessible (up, top) and less accessible (down, bottom) regions upon MDA5 expression. Data is analysed using a Hypergeometric test and the p-value adjusted using the Benjamini-Hochberg method. c Differentially expressed genes (RNA-seq) at t = 4, 8 and 24 h after Ifih1 induction were used to compute significant similarities with loss-of-function (LOF) datasets for transcription factors (TFs) in mESCs. d RT-qPCR analyses of pluripotency genes during Ifih1 induction. Data represent the average of three biological replicates ± SD. One-way ANOVA was used to calculate significant differences amongst comparisons, followed by an F-test for variance and Tukey HSD. ^*p-val ≤ 0.05, ^**p-val ≤ 0.01, ^***p-val ≤ 0.001. Refer to Source Data file for exact p-values. e Western blot analysis of KLF4 and NANOG protein levels in two independent biological replicates (1, 2) of uninduced MDA5(1) ESCs (-dox) and doxycycline treated for 14 h (+dox). Tubulin and GAPDH serve as loading controls. f ChEP-MS analyses of Ifih1 induction with the number of significantly differentially enriched proteins for each timepoint indicated (two-sided Student’s t-test, P  <  0.05, FC  >  2, 2 replicates). g KEGG pathway analyses for differentially enriched chromatin-associated proteins by ChEP-MS (two-sided Student’s t-test, P  <  0.05, FC  >  2) at 24 h and 48 h. No significant terms were obtained at 8 h. h Comparison of the differential chromatin association of key regulators of pluripotency, DNA methylation and polycomb repressive complex upon MDA5 overexpression in mESCs (n  =  2. Data are presented as the average ± s.e.m.). i Correlation between differential chromatin enrichment levels (ChEP-MS at 48 h) and RNA steady-state levels (at 8 h) upon MDA5 induction. Spearman rank correlation coefficient; ρ = 0.46, p = 0.003. j Reactome analysis of genes plotted in (i), statistics as in (b).